1. Field of the Invention
The present invention relates to a method for inactivating viruses, which is necessary for the preparation of protein formulations. The method involves adding arginine to a protein formulation at a more moderate pH than acid pH treatments used in convention methods for virus inactivation.
2. Brief Description of the Related Art
During the manufacture of protein formulations, contamination by viruses may occur. Therefore, it is absolutely necessary to provide a step in the manufacturing process of inactivating and/or eliminating viruses (ICH Harmonized Tripartite Guideline: Viral Safety Evaluation of Biotechnology Products Derived from Cell lines of Human or Animal Origin).
A plurality of treatments based on different mechanisms has been conventionally employed to inactivate viruses. These treatments include, for example, pasteurization with continuous heat (about 60° C.) for about ten hours, exposing the protein formulation to a solvent/detergent (S/D) designed for virus inactivation, such as an organic solvent such as tris-(n-butyl)-phosphate (TNBP), or the like, and a surfactant such as Tween-80 or the like, exposing the protein formulations to particular chemical substances, such as an organic acid, for example, caprylic acid or the like, an alcohol having 4 to 10 carbon atoms, β-propiolactone, and the like, treatment of the protein formulation with a photosensitive compound such as psoralen, or the like, and ultraviolet irradiation treatment of the protein formulation with gamma irradiation, and so on (Sofer, et al. BioPharm International, Oct. 42-51, 2002).
However, the target proteins are at risk of being denatured or decomposed under the severe environmental conditions created by any of the above-mentioned inactivation methods. Furthermore, when an inactivating agent is added to the protein formulation, the inactivating agent must be separated and removed from the protein formulation after the inactivation step.
Certain viruses coated with a lipid envelope are known to drastically lose their infectivity simply by being exposed to an acidic pH at low temperatures for a short period of time (0.5 to one hour). In light of this, inactivating viruses using an acid treatment with an acid such as citric acid or the like has been introduced in the production process of various kinds of protein formulations (Brorson, et al. Biotechnology and Bioengineering 82, 321-329, 2003). The above-mentioned inactivation method is a remarkably simple process for virus inactivation. To be more specific, the protein solution is adjusted to pH 5 or less using a buffer solution which acts to adjust the pH, and the protein formulation is then maintained at a chosen temperature ranging from about 0 to about 30° C. for a short period of time, and the inactivation reaction proceeds. Once the formulation is neutralized using a base, the production process of the formulation can be reinitiated. In this case, virus inactivation is triggered merely by the acid pH treatment, so no further particular chemical substances are needed. Accordingly, the extra step of removing such chemical substances is not required. Although the acid sensitivity varies among viruses, previous reports have revealed that the exposure to acidic conditions greater than pH 3.5-4 is required to effectively inactivate the viruses (Sofer, et. al. BioPharm International, Apr. 42-68, 2003; Burstyn, et al. Developments in Biological Standardization 88, 73-79 (1996)). For example, Louie et al. demonstrated how to inactivate the bovine viral diarrhea virus (BVDV) by treating with acid, and consequently found that the BVDV cannot be completely inactivated even when subjected to a pH of 4.25 at a temperature of 21° C. for 21 days (Louie, et al. Biologicals 22, 13-19, 1994). Some protein formulations use more hardy proteins that do not denature or decompose even when exposed to strongly acidic pH conditions while other formulations use proteins such as antibodies which are prone to denaturation or association under strongly acidic conditions (Paborji, M. et al: Pharmaceutical Research. 11, 764-771 (1994)). Therefore, in order to assure the quality of the target proteins, due consideration must be given regarding the acid treatment of the proteins that may cause denaturation or association under the strongly acidic conditions. Thus, there is a demand for a virus inactivation method which uses more moderate acid treatment, for example, under slightly acidic pH conditions, and which is capable of inactivating the viruses similar to the methods using stronger acids.
Milton et al. found a method for inactivating viruses when preparing immunoglobulin formulations which combines treating with solvent/detergents and treating with an acid (EP0523406). This method is conducted at pH 4 to 4.85, and greatly improves the efficiency of viral inactivation when compared with conventional solvent/detergent methods. Although this combination method can result in more moderate pH conditions, it is necessary to remove the added organic solvent and surfactant. Juergen et al. found a method for preparing immunoglobulin formulations substantially free from viruses which employs the step of inactivating the viruses by exposing the protein formulation to caprylic acid or heptanoic acid at pH 4.6 to 4.95 (WO2005082937). Johnston et al. demonstrated that the titer of BVDV was decreased to 1/10000 when the formulation was exposed to 16 mM caprylic acid at 30° C. for 10 hours and pH 4.5, and then proposed inactivating using an acid treatment and a chemical substance in combination (Biologicals 31, 213-221 (2003)). However, these methods still require the subsequent step of removing caprylic acid or the like, although more moderate pH conditions are achieved. In addition, it has been known for more than 40 years that the viruses in protein formulations can be effectively inactivated when a slight amount of pepsin is added to the protein formulation and adjusted to pH 4.0 (Jensch, et al. Transfusion 31, 423-427 (1991); Kempf, et al. Transfusion 36, 866-872 (1996)). Although more moderate pH conditions are possible in this method, the intentional addition of pepsin, for example, a protease, to the target protein is not considered to be an advantageous choice to ensure the quality of the target protein.
Arginine is known to inhibit nonspecific association and aggregation of proteins, and also is known to elute the protein from the column in purification and analysis using column chromatography (Tsumoto, et al Biotechnology Progress. 20, 1301-1308 (2004)).